The long-term goal of this proposal is to develop therapeutic strategies for the treatment of a human disease characterized by pathological changes in cellular cholesterol metabolism, Niemann-Pick type C (NPC) disease. NPC disease is a fatal neurodegenerative disorder that occurs due to mutations within a key gatekeeper protein in cholesterol transport: lysosomal Niemann-Pick type C1. Neurons from NPC patients have dramatically altered cholesterol homeostasis due to reduce transport of cholesterol out of lysosomes to the endoplasmic reticulum. Neurologically, accumulation of lysosomal cholesterol in NPC patient neurons, gives rise to impaired motor functions, psychiatric problems, seizures, dementia, and typically death prior to adulthood. Despite clear neuropathological consequences for cholesterol dysregulation in NPC disease, there has been little research attention invested in understanding whether altered ion channel function, neuronal excitability, and calcium handling, contribute to the neuropathology of NPC disease. Given that phosphoinositides control a wide range of cellular processes including the regulation of plasma membrane ion channel activity, it follows that any interdependence in the membrane pools of these lipids could have critically important implications for this disease. Our central hypothesis is that regulated efflux of cholesterol from lysosomes to the ER, via NPC1, is a rheostat for phosphoinositides and consequently neuronal excitability. To test this hypothesis, we implement a multi-scale approach to rigorously investigate the mechanisms by which cholesterol can regulate the abundance and distribution of phosphoinositides to alter phosphoinositide- dependent ion channel function. Specific Aim 1 will test the hypothesis that alterations in NPC1 function modifies plasma membrane phosphoinositides, to alter ion channel activity, and consequently neuron membrane excitability and function. In Specific Aim 2 we test the hypothesis that alterations in excitability and membrane cholesterol controls the ability of neurons to store and release calcium. Finally, in Specific Aim 3 we will determine specific proteins that have their expression profiles altered in NPC to modify phosphoinositide-dependent ion channel function. The proposed studies have specific relevance in the fields of neuroscience, cell biology and biophysics, but the fundamental importance of phosphoinositides and cholesterol for a plethora of cellular events, mean it will have broad implications for medicine.